With this phase I submission, IsoPlexis proposes to deliver comprehensive profiling of the immune system as a measure of age-dependent health in response to medical challenges or trauma. This assay will connect signaling events in the metabolome and proteome from thousands of single cells in parallel enabling the simultaneous tracking of metabolic activity and protein signaling within each immune cell type. For the purposes of improved health profiling, therapeutic targeting, and biomarkers for patient monitoring, we have received incoming demands for this novel assay from translational centers and biopharma that are focused on the aging immune system. In the normal aging process, the relative abundance of nave T cells decreases while highly differentiated effector and memory T cells increase. Functional genetic analysis of whole blood indicates global changes in immune gene signaling as well as metabolic functioning as a consequence of age. However, deciphering these changes into meaningful impact on human health in the aging immune system requires higher resolution analyses. IsoPlexis has exclusively licensed the published, underlying metabolomics technology from our collaborator and scientific advisor Jim Heath who devised a methodology to combine detection of metabolites with proteomic profiling of intracellular signaling pathways and cytokine production in single cells. The Heath lab established uniquely combined metabolite competition capture assays with sensitive signal transduction protein capture, to help uncover unique pathways and potential treatment targets. Understanding that this development has broad human health application, cell-specific metabolic and proteomic markers as a function of age- calibrated immune system performance will guide patient response to improved personalized therapies. A new general immune health assay is needed that can (i) make highly multiplexed measurements from single cells (ii) while being able to gather both proteomic and metabolic information from the same cell (iii) in a manner that is high throughput. We propose to use this phase I grant to allow IsoPlexis to develop an integrated, automated single-cell metabolomic/proteomic platform, as a comprehensive immune health assessment to better calibrate new personalized medical treatments. Aim 1: Develop and optimize an expanded metabolite capture library on- chip and integrate with intracellular proteomic assays targeting the immune system as it matures. Aim 2: Develop methods on automated platform for monitoring single-cell metabolomic/proteomic biomarkers responding to environmental stressors to evaluate immune system metabolic resilience. Transition to phase II: To demonstrate the combined metabolomic/proteomic assay value, we will evaluate a sampling from an ethnically diverse, genetically characterized pool that our collaborators at Columbia University are collecting to advance unique metabolomic and proteomic immune profiles. Additionally, the multiplexed metabolomic/proteomic assay will be integrated onto our automated platform for commercial use.